Hyperactivation of the Human Plasma Membrane Ca2+ Pump PMCA h4xb by Mutation of Glu99 to Lys

Mazzitelli, Luciana R.; Adamo, Hugo P.

doi:10.1074/jbc.m113.535583

Cited by 7 publications

(17 citation statements)

References 41 publications

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“…Limited proteolysis is a well characterized strategy to gain information on the structure of proteins at a relatively low resolution and has been extensively used to advance the knowledge of different P-ATPases [29][30][31][32][33][34][35][36][37][38][39][40][41][42]. While the cleavage sites of each protease have specific primary sequence constrains, the precise location where the cuts occur are mostly determined by the segmental exposure and flexibility [27,28].…”

Section: The Structure Of Spf1p Detected By Limited Proteolysismentioning

confidence: 99%

“…Limited proteolysis of P-ATPases has been a particularly useful technique for the study of the topology, domain function and conformational changes of P-ATPases [29][30][31][32][33][34][35][36][37][38], and has revealed the presence of non-essential regulatory domains in several members of the family, i.e. the auto-inhibitory domains of the plasma membrane Ca 2+ pumps (PMCA), the yeast plasma membrane H + -ATPase (Pma1p) and the lipid flippase Drs2p [39][40][41][42].…”

Section: Introductionmentioning

confidence: 99%

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Highly exposed segment of the Spf1p P5A-ATPase near transmembrane M5 detected by limited proteolysis

et al. 2021

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The yeast Spf1p protein is a primary transporter that belongs to group 5 of the large family of P-ATPases. Loss of Spf1p function produces ER stress with alterations of metal ion and sterol homeostasis and protein folding, glycosylation and membrane insertion. The amino acid sequence of Spf1p shows the characteristic P-ATPase domains A, N, and P and the transmembrane segments M1-M10. In addition, Spf1p exhibits unique structures at its N-terminus (N-T region), including two putative additional transmembrane domains, and a large insertion connecting the P domain with transmembrane segment M5 (D region). Here we used limited proteolysis to examine the structure of Spf1p. A short exposure of Spf1p to trypsin or proteinase K resulted in the cleavage at the N and C terminal regions of the protein and abrogated the formation of the catalytic phosphoenzyme and the ATPase activity. In contrast, limited proteolysis of Spf1p with chymotrypsin generated a large N-terminal fragment containing most of the M4-M5 cytosolic loop, and a minor fragment containing the C-terminal region. If lipids were present during chymotryptic proteolysis, phosphoenzyme formation and ATPase activity were preserved. ATP slowed Spf1p proteolysis without detectable changes of the generated fragments. The analysis of the proteolytic peptides by mass spectrometry and Edman degradation indicated that the preferential chymotryptic site was localized near the cytosolic end of M5. The susceptibility to proteolysis suggests an unexpected exposure of this region of Spf1p that may be an intrinsic feature of P5A-ATPases.

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Section: The Structure Of Spf1p Detected By Limited Proteolysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Highly exposed segment of the Spf1p P5A-ATPase near transmembrane M5 detected by limited proteolysis

et al. 2021

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“…The purification procedure was adapted from our previously established method for the purification of pig brain PMCA [7], and gave a yield increase of at least 10-fold for the native hPMCA4b, when compared to previous methods [17,39]. Although they are similar, it can be possible that our plasmid construct gets higher expression values than those used in previously published works.…”

Section: Discussionmentioning

confidence: 95%

“…First we have assured a conveniently high expression level for the recombinant protein hPMCA4b in S. cerevisiae by the use of the strong constitutive promoter GPD enclosed in the high copy number plasmid p426GPD, while previous methods relied on inducible promoters [17,39]. Previous protocols for PMCA extraction from human or animal tissues are not suitable for S. cerevisiae since yeasts have cell wall and this complicates cell lysis.…”

Section: Discussionmentioning

confidence: 99%

An improved method for expression and purification of functional human Ca2+ transporter PMCA4b in Saccharomyces cerevisiae

Corbacho

García-Prieto

Hinojosa

et al. 2016

Protein Expression and Purification

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“…Perturbations in these processes underlie the pathogenesis of autism spectrum disorders (Gilbert and Man, 2017 ). ATP2B3 mutations are associated with X-linked cerebellar ataxia and Ca 2+ extrusion disorders in patients with cerebellar ataxia and developmental delay (Zanni et al, 2012 ; Mazzitelli and Adamo, 2014 ; Cali et al, 2015 ). Several neurotoxic agents, such as oxidation and age, downregulate PMCA function and increase susceptibility to NDs (Zaidi, 2010 ).…”

Section: Pathophysiological Role Of Plasma Membrane Calcium Atpasesmentioning

confidence: 99%

The Role of TRP Channels and PMCA in Brain Disorders: Intracellular Calcium and pH Homeostasis

Hwang

Lee

Kim

2021

Front. Cell Dev. Biol.

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Brain disorders include neurodegenerative diseases (NDs) with different conditions that primarily affect the neurons and glia in the brain. However, the risk factors and pathophysiological mechanisms of NDs have not been fully elucidated. Homeostasis of intracellular Ca2+ concentration and intracellular pH (pHi) is crucial for cell function. The regulatory processes of these ionic mechanisms may be absent or excessive in pathological conditions, leading to a loss of cell death in distinct regions of ND patients. Herein, we review the potential involvement of transient receptor potential (TRP) channels in NDs, where disrupted Ca2+ homeostasis leads to cell death. The capability of TRP channels to restore or excite the cell through Ca2+ regulation depending on the level of plasma membrane Ca2+ ATPase (PMCA) activity is discussed in detail. As PMCA simultaneously affects intracellular Ca2+ regulation as well as pHi, TRP channels and PMCA thus play vital roles in modulating ionic homeostasis in various cell types or specific regions of the brain where the TRP channels and PMCA are expressed. For this reason, the dysfunction of TRP channels and/or PMCA under pathological conditions disrupts neuronal homeostasis due to abnormal Ca2+ and pH levels in the brain, resulting in various NDs. This review addresses the function of TRP channels and PMCA in controlling intracellular Ca2+ and pH, which may provide novel targets for treating NDs.

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Hyperactivation of the Human Plasma Membrane Ca2+ Pump PMCA h4xb by Mutation of Glu99 to Lys

Cited by 7 publications

References 41 publications

Highly exposed segment of the Spf1p P5A-ATPase near transmembrane M5 detected by limited proteolysis

Highly exposed segment of the Spf1p P5A-ATPase near transmembrane M5 detected by limited proteolysis

An improved method for expression and purification of functional human Ca2+ transporter PMCA4b in Saccharomyces cerevisiae

The Role of TRP Channels and PMCA in Brain Disorders: Intracellular Calcium and pH Homeostasis

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